Vegetative propagation of agriculturally important plant species is a well known and valuable practice in some fields of agriculture. In the horticultural arts for example, ornamental flowers are vegetatively propagated from callus. Callus may be defined as a collection of plant cells that have become dedifferentiated usually under the influence of plant hormones such as auxins, for example indole acetic acid (IAA) and auxin-like compounds, for example 2,4-dichloro phenoxyacetic acid (2,4-D). Cellular division of such dedifferentiated plant cells usually produces poorly organized cells that may be further propagated in a growth medium, provided that an auxin or auxin-like plant hormone is present in the growth medium. By adjusting nutrient, vitamin, and plant hormones levels, plants that are essentially identical to the individual plant from which the callus was initiated can be regenerated. For example, using these general methods, the production of thousands of identical orchids is routinely possible from the somatic cells of a single orchid plant.
Vegetative propagation of crop plants by inducing callus, maintaining the callus and regenerating plants from the callus has great potential in the development and improvement of food crops. For example, vegetative propagation may be used to multiply inbred or hybrid plant strains that have commercially valuable or scientifically interesting characteristics which occur rarely or are unstable when the plant reproduces sexually. Tissue obtained from such scientifically interesting or commercially valuable plants can be used to propagate numerous copies of the original plant, thereby stably maintaining the otherwise unstable characteristics.
Callus induced from the tissue of a unique or valuable plant can be multiplied and plants can be regenerated from the callus. Thus multiple copies of a single unique or valuable plant can be reproduced for further breeding, genetic selection or genetic manipulation using recombinent nucleic acid techniques. Plants having unique traits can thus be used in breeding programs without risk of losing the desirable characteristics that may be unstable.
Sunflower (Helianthus annuus, hereinafter referred to as H. annuus), is one of four major crops grown for edible oil. Ninety percent of the sunflower crop grown in the United States is used for oil production. Development of a system that permits oil-producing inbreds of H. annuus to regenerate plants from tissue or callus or suspension culture would be useful as a supplement to current plant breeding programs.
Most reports on tissue culture of H. annuus have concerned crown gall cells, i.e., plant tumor cells produced as a result of infection by the bacterial plant pathogen Agarobacterium tumafaciens, but there has been some work on callus production from normal sunflower tissues (see for example, Kandler, O., Planta, Bd. 40: 346-349 (1952); Henderson et al., Amer. J. Bot. 39: 467-473 (1955); Rogers et al., In Vitro 9: 462-467 (1974). In addition, there have been three reports of plant or shoot regeneration from cultured sunflower tissue. M. K. Sadhu reported plant regeneration from sunflower stem pith cultured on a modified White's medium with 1 part per million (ppm) indole-acetic acid (IAA) (see Sadhu, M. K., Indian J. Exp. Biol., 12: 110-111 (1979)). Binding et al. reported shoot regeneration from protoplasts isolated from cultured shoot tips, although no details were presented (Binding et al. Z Planzenphysiol-Bd. 101: 119-130 (1981)). In these two reports, no information was presented regarding the seed source. The source could have been confectionary (nonoil-producing) hybrids or open pollinated varieties.
A recent report (Guco et al., 1984), also using asynthetic variety, shows regeneration using 2,4-D and BA. No mention is made of the ability to maintain this callus. Georgieva-Todorova et al. reported callus induction and shoot generation from two sterile hybrids--H. annuus.times.H. decapetalus and H. annuus.times.H. hirsutus (Georgieva-Todordva, et al. Proc. 9th Int'l Sunflower Conf. Torremolinos Spain (1980)).
Georgieva-Todorova et al. specifically disclose a medium that allow sunflower callus growth and organogenic regeneration of sunflower shoots from tissue of sterile hybrid sunflower plants. The medium disclosed by Georgieva-Todorova (GT medium) is composed of a range of plant hormones, White's vitamins, and major and minor salts disclosed in Murashige, T. and Skoog, F., (1962) Physiologia Plantarium, 15: 443-97. The vitamins disclosed in GT medium are as follows:
______________________________________ Vitamin Component Concentration (per 1 medium) ______________________________________ thiamine.HCl 0.1 mg nicotinic acid 0.5 mg pyridoxine.HCl 0.5 mg Ca--pantothenate 1.0 mg inositol 500.0 mg glycine 3.0 mg adenine sulfate 40.0 mg cysteine 1.0 mg ______________________________________
The plant hormones disclosed by Georgieva-Todorova et al. are numerous and depending upon the particular mix of hormones and concentration of the particular hormone used in the mix various results are obtained. Thus quick growth of callus with rapid differentiation of meritematic centers is observed in the GT media described above having 0.1 or 10 mg/l benzylamino purine (benzyladenine hereinafter referred to as BA); 0.1 or 10 mg/l indolbutyric acid (hereinafter IBA); 0.1 mg/l BA with 0.1 or 10 mg/l naphthalene acetic acid (NAA); and a combination of 10.0 mg/l BA with 0.1 or 10 mg/l NAA.
Specifically, the best organogenesis was observed in GT medium composed of MS salts and vitamins as described above and the plant hormones as follows: 0.1-0.5 mg/l NAA, 0.1 mg/l BA, and 0.01 mg/l gibberellic acid (GA). The best organogenesis occurred with this mix of plant hormones when the NAA concentration was 0.1 mg/l.
The experiments of Georgieva-Todovova et al. disclose several plant hormone concentrations that appear to permit significant regeneration of sunflower tissue in two sterile hybrid sunflower seed lines. As will be seen below Georgieva-Todorova et al. do not, however, disclose media that may be used to regenerate sunflower tissue from inbred sunflower seed lines. The value of the regeneration medium disclosed by Georgieva-Todovova et al. is thus limited, since the sunflower seed lines to which it has been successfully applied are sterile hybrid seed lines that cannot be used for breeding or genetic selection purposes.
One of the problems facing workers seeking to regenerate sunflower plants from inbred sunflower lines is to develop artificial culture media for callus culture and/or suspension culture (i.e., cells or small clumps of cells suspended in liquid artificial medium) of inbred sunflower lines. A further problem is to identify those inbred sunflower lines that have the potential for successful regeneration of whole plants from callus or suspension culture. The potential of any particular sunflower line to regenerate whole plants is determined by the interaction of the inherent regeneration characteristics of the sunflower line and the medium on which the regeneration of the plants is carried out. Once this regeneration potential has been identified on a sunflower line, it is desirable to develop a media optimized for stably maintaining callus with regeneration potential and for regeneration of sunflower from tissue culture.
As a practical matter, when regenerating from callus or suspensions of sunflower lines identified for their regeneration potential, it is desirable to have a method that can maintain the sunflower callus or suspensions in a condition that permits rapid and prolific regeneration when desired or convenient, yet does not commit the whole sunflower callus or suspensions sample to regeneration and consequent loss of the callus or suspensions line. Media currently used to maintain sunflower callus or suspensions in culture for extended periods of time do not permit any significant regeneration when sunflower callus or suspensions are cultured on them. However, sunflower tissue grown on such maintenance medium for extended periods generally loses the ability to regenerate because, it is believed, the known maintenance media select against regenerable cells and tissues in the callus.
Regeneration media such as that disclosed in Georgievna-Todorova et al. mentioned hereinabove and in the inventors co-pending application Ser. No. 584,079, are effective for inducing regeneration in primary explants from sunflower seedlings and thus are suitable for screening for regeneration potential. These regeneration media, however, are not suitable for maintaining regenerable sunflower callus.
In light of the limitations of conventional maintenance and regeneration media currently in use, it is desirable to develop a medium, hereinafter referred to as induction medium that will stably maintain sunflower callus or suspensions for extended periods of time, at least several months, in a readily regenerable state without causing significant sunflower regeneration. It is furthermore highly desirable to develop a medium, hereinafter referred to as rapid regeneration media, that sustains rapid proliferation and sunflower regeneration, of tissue transferred from the induction medium to the rapid proliferation medium.